Devices and methods for removing target agents from a sample

ABSTRACT

The invention provides devices, test kits and methods for removing target agents from a sample. The device contains one or more porous matrices having pore sizes larger than 10 μm, and a plurality of particles impregnated therein. The target agents attach the device and are removed from the sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 37 U.S.C. §119(e) based on U.S.Provisional Application No. 60/617,669, filed Oct. 13, 2004, whichclaims benefit under 35 U.S.C. §119(e) to U.S. Provisional ApplicationNo. 60/616,118, filed Oct. 6, 2004, which claims benefit under 35 U.S.C.§119(e) to U.S. Provisional Application No. 60/578,061, filed Jun. 9,2004, the entire contents of which are incorporated herein by reference.

I. FIELD OF THE INVENTION

This invention relates to devices and methods for removal of targetagents from a sample. In particular, the invention relates to removal ofpathogens from biological samples.

II. BACKGROUND OF THE INVENTION

The process of adsorption of biological species to solid supports findsa number of practical applications in purification, detection andremoval of target molecules from multicomponent streams. For example,ion exchange, hydrophobic and affinity ligands are able to adsorb manyagents preferentially to chromatographic supports to affect theirseparation from aqueous solutions. Once adsorbed, the biological agentcan either be eluted as a product, or detected by ELISA and otheranalytical approaches.

In some instances, the solution containing the target biological agentalso contains large entities such as red blood cells, viruses, bacteria,liposomes, leukocytes, and aggregates of various sizes. In many of theseinstances, it is desirable to allow the large aggregates to flow throughthe solid matrix or support without interfering with the ability of thetarget biological agents to bind to the support. This requires porespaces in the solid matrix that are large enough to accommodate the flowof the large entities. Unfortunately, large pore spaces can have a lowsurface area that limits the capacity of the solid matrix to bind to thetarget agents.

In other cases, it is desirable to actually filter the large particlesto facilitate adsorptive separation of the smaller target agents. Oneexample is the removal of cells from a culture medium to recover anextracellular product.

In addition, there are many instances where it is desirable to bind,rather than filter, the biological entities that are very large. Forexample, it is of importance to adsorb specifically many pathogens,including infectious prions, viruses, bacteria, and toxins from mixturesof biological agents. These entities often have difficulty accessing thesmall pores that are required for binding in the currently availablesample purification and separation devices.

Nonwoven fibers or webs, also referred to as melt blown polymer fibersor spunbonded webs, are well known and are used for filtration andseparation of fine particles from air and aqueous solutions. (see, forexample, U.S. Pat. Nos. 4,011,067 and 4,604,203, each of which isincorporated herein by reference in its entirety). Loading of sorptiveparticulates in nonwoven webs is also well known in the art (see, forexample, U.S. Pat. Nos. 4,433,024; 4,797,318; and 4,957,943, each ofwhich is incorporated herein by reference in its entirety). Applicationsinclude face respirators for removing particulates and gaseouscontaminants, protective garments, fluid retaining articles, and wipersfor oil.

More recently, methods for the fabrication of particle impregnatednonwoven fabrics for separation and purification have been reported.See, for example, U.S. Pat. No. 5,328,758, incorporated herein byreference in its entirety. The patent teaches functionalized particlesfor the attachment of affinity ligands. It is disclosed that theparticles are blown into the polymer fibers during the melt blowingstage. The nonwoven fabric comprises pores having pore sizes in therange of 0.24 to 10 μm, preferably 0.5 to 5 μm. It is also specifiedthat the impregnated fabric material must have a Gurley Time of at least2 seconds.

WO93/01880 discloses a leukocyte-removing nonwoven fabric filtermaterial produced by dispersing in a medium a mass of a great number ofsmall fiber pieces having a fiber diameter of not more than 0.01 μm anda length of about 1 to 50 μm, together with spinable and weavable shortfibers having an average length of 3 to 15 mm. U.S. Pat. Nos. 4,550,123and 4,342,811, each of which is incorporated herein by reference in itsentirety, describes microporous polymeric fibers and films which containparticles capable of sorbing vapors, liquids, and solutes. Typicalsorbent particles include active carbon, silica gel, and molecularfilter type materials.

The invention as disclosed herein provides devices and methods forsample purification, and detection and removal of target agents from asample with increased efficiency and specificity and substantial savingsin time and cost over the devices of the prior art.

III. SUMMARY OF THE INVENTION

The invention, as disclosed and described herein, provides methods,devices and kits for removing target agents from a sample.

In one aspect, the invention provides a device for separating at leastone target agent from a sample. The device contains one or more porousmatrices having pore sizes larger than 10 μm, and a plurality ofparticles impregnated therein, wherein the at least one target agentattaches to the one or more porous matrices, particles, or both and isremoved from the sample. In one embodiment, the porous matrix, theparticles or both have uniform or variable pore sizes. In anotherembodiment, the particles have a pore size of about 0.001 μm to about0.1 μm. In yet another embodiment, the particles comprise a porous resinhaving interconnected pores with surface areas in the range of about 1-2m²/g of dried resin to about 300 m²/g of dried resin.

In yet another embodiment, the porous matrix comprises natural fibers,synthetic fibers or both. In a preferred embodiment, the porous matrixcomprises at least one nonwoven fabric. In another embodiment, theporous matrix is a blend of two or more of the same or different typesof woven and/or nonwoven fabrics.

In yet another embodiment, the device of claim 1, wherein the particlescomprise a polymethacrylate, a methacrylate resin, a modified resin, ora combination thereof.

In one embodiment, the device contains a modified resin and one or moreporous matrices comprise plasma treated polypropylene that isfunctionalized with a reactive group comprising a ligand having aprimary amine and a hydrophilic spacer containing polyethylene glycolunits.

In another embodiment, the particles are sandwiched between the one ormore porous matrices.

In one embodiment, the particles, the porous matrix or both arefunctionalized with one or more reactive groups. The target agents areattached to the particles, porous matrix or both via absorption,adsorption, ion exchange, covalent bonds, hydrophobic, dipole,quadrupole, hydrogen bonding, specific interactions, formation ofcharged species, via affinity interaction to specific ligands, or acombination thereof. In yet another embodiment, the particles arepolymethacrylate or a methacrylate resins including, by way of exampleand not limitation, a FRACTOGEL™ EMD, a TOYOPEARL™, or a TSK-GEL™polymer matrix. In yet another embodiment, the resin is TOYOPEARL™ Amino650 including, for example, Amino 650 U, Amino 650 M, or a partialacetylated form of the Amino 650M or Amino 650 U. Partial acetylatedresin includes from about 5% to about 95% or more acetylated resins. Inone embodiment, partial acetylated resin includes from about 10% toabout 85% acetylated resin. In another embodiment, partial acetylatedresin includes from about 20% to about 75% acetylated resin. In yetanother embodiment, partial acetylated resin includes from about 30% toabout 60% acetylated resin. In another embodiment, partial acetylatedresin includes from about 40% to about 60% acetylated resin. It isintended herein that by recitation of such specified ranges, the rangesrecited also include all those specific integer amounts between therecited ranges. For example, in the range about 40 and 60%, it isintended to also encompass 45%, 50%, 55%, 57%, etc, without actuallyreciting each specific range therewith. In another embodiment, the resinincludes wet resins (i.e., fully pre-hydrated), dry resins (i.e., notpre-hydrated before contact with the sample, and/or previously dry buthydrated before contact with the sample). The use of a partialacetylated dry and/or wet resin is also encompassed within the scope ofthe invention.

In another aspect, the device contains a functionalized porous nonwovenor woven matrix that has the ability to adsorb the target agents. In oneembodiment, the device contains a nonporous matrix as well as porousmatrix, one or both of which matrices may be functionalized. In anotherembodiment, the porous matrix contains uniform or variable pore sizeslarger than 10 μm.

In yet another aspect, the invention provides methods of separating atleast one target agent from a sample comprising; (a) providing a samplepotentially containing one or more target agents; (b) providing a devicecomprising (i) one or more porous matrices having pore sizes larger than10 μm, and (ii) a plurality of particles impregnated in the porousmatrix, wherein the particles have the capacity of attaching at leastone target agent; (c) subjecting the sample to the device; (d) attachingat least one target agent to the particles, to the one or more porousmatrices or both; and (e) separating at least one target agent from thesample.

In another aspect, the invention provides test kits for targetseparation, detection and sample purification comprising one or more ofthe following (i) a device containing a porous matrix having pore sizeslarger than 10 μm, and a plurality of particles impregnated therein,(ii) a container containing one or more of buffers, reagents, chemicalagents, functionalization reagents, enzymes, detection agents, controlmaterials, (iii) instructions for use of the test kit, and (iv)packaging materials.

Other preferred embodiments of the invention will be apparent to one ofordinary skill in the art in light of what is known in the art, in lightof the following drawings and description of the invention, and in lightof the claims.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Depicts a schematic representation for resin impregnated nonwovenfabrics (RINs). The nonwoven fabrics have a pore size of about 12 μm andare impregnated with a porous resin support. The mean pore size issufficiently large to allow red blood cells to flow freely through thedevice without exhibiting any signs of damage. Particles (10), fibers(20), impregnated particles (11), and nonwoven fabric (21) are showntherein.

FIG. 2 Depicts a schematic representation of a device composed of squaresheets of a nonwoven or woven fabric. A staggered array of sheets ofnonwoven or woven fabric is coated with affinity ligands on both sides.The sample flows in the tortuous path between the sheets. The pore sizeof the sheets is adjusted according to the desired application.

FIG. 3 Depicts a scanning electron micrograph of sample 13 inner/innerlayer calendered at 150 F, and 100 pounds per linear inch (PLI) withresin. The micrograph shows pocket areas of sample 13 at 50×magnification.

FIG. 4 Depicts a scanning electron micrograph of sample 11 inner/outerlayer calendered at 180 F, and 400 PLI, with resin. The micrograph showspocket areas of sample 11 at 50× magnification.

FIG. 5 Depicts a bargraph indicating the results of a Micro BCA assay of12 fractions collected for the different number of membrane (porousmatrix) layers. Different β-Lactoglobulin concentrations were showed onflow-through fractions of a solution passing through 1 layer ofresin-embedded membrane (labeled “resin”) or one layer of membrane(labeled “control”).

FIG. 6 Depicts a bargraph indicating the results of a Micro BCA assay of12 fractions collected for the different number of membrane layers.Different β-Lactoglobulin concentrations were showed on flow-throughfractions of a solution passing through 2 layers of resin-embeddedmembrane (labeled “resin”) or 2 layers of membrane (labeled “control”).

FIG. 7 Depicts a bargraph indicating the results of a Micro BCA assay of12 fractions collected for the different number of membrane layers.Different β-Lactoglobulin concentrations were showed on flow-throughfractions of a solution passing through 3 layers of resin-embeddedmembrane (labeled “resin”) or 3 layers of membrane (labeled “control”).

FIG. 8 Depicts a bargraph indicating the results of a Micro BCA assay of12 fractions collected for the different number of membrane layers.Different β-Lactoglobulin concentrations were showed on flow-throughfractions of a solution passing through 4 layers of resin-embeddedmembrane (labeled “resin”) or 4 layers of membrane (labeled “control”).

FIG. 9 Depicts distribution of particles on membrane rolls. Resinparticles were dispersed uniformly onto the bottom membrane with nospilling over the edges of the membrane.

V. DETAILED DESCRIPTION OF THE INVENTION

Methods, devices and kits for efficient separation of target moleculesfrom a sample are described herein. The methods, kits and devices of theinvention are useful in a variety of applications includingpurification, separation, and processing of expressed gene products fromcells, production and delivery of biopharmaceuticals, and prognostic,diagnostic, and/or detection applications, among others. The inventiondescribed herein defines novel devices that separate differentcomponents of a sample and allow the flow of larger species through thedevice while providing large surface areas to bind the target agents.

Particular applications of this invention involve the removal ofpathogens such as prions, viruses, fungus, bacteria and toxins from abiological samples such as, for example, a blood sample including wholeblood, red blood cell containing compositions, red blood cellconcentrates, platelets concentrates, plasma, plasma derivatives,leukocytes, leukodepleted blood, mammalian cell culture, fermentationbroths and other media used for the manufacture and delivery ofbiopharmaceuticals and the preparation of therapeutics.

1. Definitions

The definitions used in this application are for illustrative purposesand do not limit the scope of the invention.

As used herein, “modified resins” are defined broadly within the scopeof the invention and include analogues, variants and functionalderivatives of a resin with or without a functional group. Themodification includes for example, substitution, deletion, or additionof chemical entities (e.g., amino acids) to a particular resin, or itsfunctional group, or both. For example, amino substitution, acetylation,and/or partial acetylation of resins are included in the definition ofmodified resins.

As used herein, “target agents” are defined broadly within the scope ofthe invention and include chemical, biological, or physical agents thatare captured by the device of the invention. Target agents includemolecules, compounds, cell constituents, organelles, aggregates, toxin,prions, and microorganisms such as pathogens including, virus, bacteria,fungi, and protozoa, among others. Target molecules also includepolymeric molecules such as polynucleotide molecules, for example, DNA,RNA, DNA-RNA hybrid, antisense RNA, cDNA, genomic DNA, mRNA, ribozyme,natural, synthetic, or recombinant nucleic acid molecules,oligopeptides, oligonucleotides, peptides, peptide-nucleic acid hybrids,antigen, antibody, antibody fragments, large proteins and aggregatessuch as vWF:FVIII, and HDL among others.

As used herein, the term “pathogen” is intended to mean any replicableagent that can be found in or infect a biological sample such as a bloodsample. Such pathogens include the various viruses, bacteria, protozoa,and parasites known to those of skill in the art to generally be foundin or infect whole blood or blood components and other pathogeniccontaminants not yet known. Illustrative examples of such pathogensinclude, but are not limited to, bacteria, such as Streptococcusspecies, Escherichia species and Bacillus species; viruses, such ashuman immunodeficiency viruses and other retroviruses, herpes viruses,paramyxoviruses, cytomegaloviruses, hepatitis viruses (includinghepatitis A, hepatitis B, and hepatitis C viruses), pox viruses and togaviruses; and parasites, such as malarial parasites, including plasmodiumspecies, and trypanosomal parasites.

As used herein, “sample” includes any sample containing a target agentthat can be captured by the device and method of the invention. Samplesmay be obtained from any source that potentially contains a targetagent. Such sources include animals, plants, soil, air, water, fungi,bacteria, and viruses, among others. Animal samples are obtained, forexample from tissue biopsy, blood, hair, buccal scrapes, plasma, serum,skin, ascites, plural effusion, thoracentesis fluid, spinal fluid, lymphfluid, bone marrow, respiratory fluid, intestinal fluid, genital fluid,stool, urine, sputum, tears, saliva, tumors, organs, tissues, samples ofin vitro cell culture constituents, fetal cells, placenta cells oramniotic cells and/or fluid, among others.

As used herein, “cell culture media” includes any prokaryotic oreukaryotic culture media such as, for example, bacterial, yeast andother microbiological cell culture media, mammalian cell culture media,plant cell culture, and insect culture, fermentation broths and othermedia used for the production and delivery of biopharmaceuticals and thepreparation of therapeutics.

As used herein, “blood sample” includes, for example and not by way oflimitation, whole blood, red blood cell-containing compositions (e.g.,red blood cell concentrates and platelets concentrates), leukocytes, andleukodepleted blood, blood proteins, such as blood clotting factors,enzymes, albumin, plasminogen, and immunoglobulins; and liquid bloodcomponents, such as plasma, plasma derivatives, and plasma-containingcompositions among other blood samples.

As used herein, the term “red blood cell-containing composition” meanswhole blood, red blood cell concentrates and any other composition thatcontains red blood cells. Other than red blood cells, the compositioncan also contain a biologically compatible solution, such as ARC-8,Nutricell (AS-3), ADSOL (AS-1), Optisol (AS-5) or RAS-2 (Erythrosol),and one or more cellular blood components, one or more blood proteins,or a mixture of one or more cellular blood components and/or one or moreblood proteins. Such compositions may also contain a liquid bloodcomponent, such as plasma.

As used herein, “particle” means organic or inorganic porous ornonporous forms having a diameter of about 1 to about 200 μm or more,these include, for example and not by way of limitation, fibers with alength to diameter ratio of about 1 μm to about 20 μm or more, inaddition to sorptive particles such as granules, beads, resins, orpowders, among others.

As used herein, “sorbent” “sorptive” or “sorption” means capable oftaking up and holding by either absorption or adsorption.

As used herein, “attachment” is broadly defined within the scope of theinvention and includes any type of physical, chemical, or biologicalbonding processes between two entities and includes, for example and notby way of limitation, absorption, adsorption, covalent bonding, ionexchange, hydrophobic, hydrogen bonding, dipole, quadrupole or affinityinteraction, formation of charged species, the attachment of affinityligands (e.g., including peptides, oligonucleotides, proteins, spacerarms, hydrophobic moieties, fluorinated materials), among others.

As used herein, “spiked solution” refers to a solution that has receiveda certain amount of the target protein, toxin, virus, bacteria, or otherorganism, in its pure, partially purified, or crude form.

2. Porous Matrix

The devices of the present invention comprise a porous matrix havingparticles impregnated therein. Selection of a porous matrix can varywidely within the scope of the invention. Useful matrices include wovenand nonwoven fabrics (such as fibrous webs), microporous fibers, andmicroporous membranes. These fibers can be made out of any materials andany methods known to the art, including meltblowing, spinbonding, andelectrospinning.

Fibrous webs are particularly desired because such webs provide largesurface areas, with nonwoven fibrous webs being preferred due to ease ofmanufacture, low material cost, and allowance for variation in fibertexture and fiber density. A wide variety of fiber diameters, e.g., 0.05to 50 μm, is used in the preparation of the device of the presentinvention. The matrix thickness is varied to fit the desired utility ofthe device, e.g., about 0.1 μm to about 100 cm thick or more. The matrixcan be used in the form of a single sheet or stacked as desired toachieve the desired capacity for adsorption. In one embodiment,calendering or pressurizing of the porous matrix is required in order toachieve the desired thickness and pore size. The porous matrix of thedevices of the invention is made from a wide variety of natural andsynthetic fibers, according to the precise physical and chemicalproperties of the porous matrix intended for the end application. Theporous matrix of the invention is selected from natural or syntheticsources including, for example, polyester, polypropylene, rayon, aramid,and/or cotton, among others.

Also encompassed within the scope of the present invention is the use oftwo or more different matrices with different chemical or physicalcharacteristics. In one embodiment of the present invention, the porousmatrix is a blend of two or more of the same or different types of wovenand/or nonwoven fabrics. In another embodiment, a hybrid of two or moreporous matrices with different pore sizes is used, one matrix havingsmaller pore sizes acts to capture the smaller materials whereas theother matrix having larger pore sizes acts as a filter for largermaterials (leukocytes for example). In another embodiment, afunctionalized porous matrix for affinity separations having apredetermined pore size is placed within another membrane as a support.

2.1. Nonwoven Fabrics

Nonwoven fabrics are random fibrous webs, formed by mechanical, wet orair laid means and having interconnecting open areas through the crosssection. Nonwoven fabrics are usually flat, porous sheets that are madedirectly from separate fibers or from molten plastic or plastic film.These fabrics are broadly defined as sheet or web structures bondedtogether by, for example, entangling fiber or filaments or perforatingfilms mechanically, thermally or chemically using various techniquesincluding adhesive bonding, mechanical interlocking by needling or fluidjet, entanglement, thermal bonding, and stitch.

Typically, nonwoven fabrics have mean pore flow (MPF) diameters rangingfrom about 1 to about 500 μm. In one embodiment, the porous matrix has apore size of at least 10 μm. In a preferred embodiment, the porousmatrix has a pore size of more than 10 μm. In yet another embodiment,the porous matrix has a pore size of more than 15 μm. It is intendedherein that by recitation of such specific numerical values, the valuesrecited also include all those specific integer amounts between therecited values. For example, more than 10 μm is intended to alsoencompass 12, 20, 30, 45, 70, 100, 200, 300, 400 and 500 μm, etc.,without actually reciting each specific range therein.

The mean pore diameter of the fabric can be chosen to correspond to thedesired pore diameter for flow of the large aggregates in the biologicalmixture. For example, in the case of red blood cells, pore flow diameterwould be on the order of about 12 μm. In this case, any porous ornon-porous particle with diameters much beyond 12 μm would be trapped inthe spaces between the fibers and particles would still be available foradsorption of the target species. As a result, significantly smallerdiameter particles can be used for adsorption while allowing flow of thelarger species through the pore spaces.

Nonwoven fabrics are made of fibers that, depending on the fabricationmethod, have diameters in the range of, for example, from about 0.01 toabout 10 μm. The fibers consist of a wide variety of materials includingnatural fibers and synthetic fibers. Natural fibers include, forexample, cellulose, cotton, and wool, among others. Synthetic fibersinclude common polymers such as polypropylene and polyester (PET,polyethylene terephthalate). Suitable polymers include polyalkylenessuch as polyethylene and polypropylene, polyvinyl chloride, polyamidessuch as the various nylons, polystyrenes, polyarylsulfones, polyvinylalcohol, polybutylene, ethyl vinyl acetate, polyacrylates such aspolymethyl methacrylate, polycarbonate, cellulosics such as celluloseacetate butyrate, polyesters such as poly (ethylene terephthalate),polyimides, and polyurethanes such as polyether polyurethanes, andcombinations thereof.

Nonwoven fabrics can also be prepared from combinations of co-extrudedpolymers such as polyester and polyalkylenes. Copolymers of the monomersof the polymers described above are also included within the scope ofthe present invention. Additionally, nonwoven fabrics are combined webswhich are an intimate blend of fine fibers and crimped staple fibers. Inone embodiment, the nonwoven fabric of the device of the invention alsoincludes a permeable support fabric laminated to one or both sides ofthe fabric, as described in U.S. Pat. No. 4,433,024 (incorporated hereinby reference in its entirety), or additionally contains reinforcingfibers.

Nonwoven fabrics are made by different means, including meltblowing andspinbinding. There are several mechanical approaches to bonding nonwovenfabrics together, for example, membranes are welded together using anultrasound cutter/sealer or by the use of a press to apply heat andpressure simultaneously. Dry-laid nonwovens contain layers of fibers,each layer containing randomly positioned or parallel fibers. Bondingwith an adhesive or heat is necessary for the dry-laid nonwoven fabric.Wet-laid nonwoven fabrics are paper-like nonwovens containing a randomarray of layered fibers, with the layering resulting from the depositionof fibers from a water slurry. Needlepunched nonwoven fabrics arecharacterized by the entangled condition of fibers of which they arecomposed, with the entanglement resulting from the application of heat,moisture and agitation to a fibrous web. Spunlaced nonwoven fabrics havefibers entangled by action of high-velocity water jets (process alsocalled hydroentanglement).

3. Particles

In one aspect, the device of the present invention includes particles aswell as the porous matrix. Particles have a capacity to attach thetarget agents. The particles are porous, non porous or both. In oneembodiment, the porous particles are sorbent particles capable ofadsorption or absorption of the target agent. The particles are made ofone material or a combination of two or more materials, which materialsare non-swellable or swellable in organic fluids or aqueous fluids andare substantially insoluble in water or fluids. It has been foundadvantageous in some instances to employ particles in two or moreparticle size range falling within the broad range.

Size and shape of the particles can vary widely within the scope of theinvention and depend to some extent upon the type of porous matrixsupport into which such particles are incorporated. For example,particles have a spherical shape, a regular shape, or an irregularshape, or a combination thereof.

Particles used in the device of the invention have an apparent sizewithin the range of about 1-2 μm to about 200-300 μm. In general,differences in useful particle sizes are dictated by the type of theporous matrix in which particles are incorporated, processes andequipment which are utilized to form the porous matrix and the porosityof the matrix so formed. For example, nonwoven fibrous webs andfibrillated polymer matrices can be formulated with the entire sizerange of particles. Preferably, about 40-200 μm sized particles are usedfor the nonwovens while 1-100 μm sized particles are preferred forfibrillated polytetrafluoroethylene (PTFE) matrices.

Also included within the scope of the present invention are particleshaving a wide range of pore sizes. Particles with a relatively largepore size are used for the efficient capture of the larger targetmolecules, such as proteins, while particles with smaller pore sizes areused for the efficient capture of smaller target molecules. The range ofavailable pore sizes is for example, from about 0.001 μm to about 0.1μm. In one embodiment, the pore sizes are about 0.1-0.55 μm. In anotherembodiment, the pore sizes are about 0.6-2 μm. In yet anotherembodiment, the pore sizes are about 0.25-5 μm or more. It is intendedherein that by recitation of such specified ranges, the ranges recitedalso include all those specific integer amounts between the recitedranges. For example, in the range of about 0.1-0.55 μm, it is intendedto also encompass 0.2, 0.3, 0.4, 0.5 μm etc, without actually recitingeach specific range therewith.

The particles are made of carbon or an organic compound which can be apolymer or copolymer. For example, particles are made of a copolymer ofstyrene and divinylbenzene and derivatives thereof, polymethacrylateester, derivatized azlactone polymer or copolymer, organic coatedinorganic oxide particles such as silica, alumina, aluminum oxide,titania, titanium oxide, zirconia, and other ceramic materials, glass,cellulose, agarose, and a wide variety of different polymers, includingpolystyrene and polymethylmethacrylate, acrylic resins and and othertypes of gels used for electrophoresis, among others.

Other suitable particles for the purposes of this invention include anyparticle which can be coated with insoluble, swellable, or non-swellablesorbent materials on their external and/or internal surfaces. In oneembodiment, the particles swell to a volume of about 2-5 times or moreas compared to their original dry weight.

The function of coating is to provide specific functionalities andphysical properties, which can be tailored according to the specificseparation assay intended. These functions include sorption, ionexchange, chelation, steric exclusion, chiral, affinity, etc. Preferredparticle material for such coatings includes inorganic oxide particles,most preferably silica particles. Such particles having coated surfacesare well known in the art, see, for example, Snyder and Kirkland,“Introduction to Modern Liquid Chromatography”, 2d Ed., John Wiley &Sons, Inc. (1979) and H. Figge et al., Journal of Chromatography 351(1986) 393-408 and include modified silica particles, silica particleshaving covalently bonded organic groups including cyano, cyclohexyl, C₈(octyl), and C₁₈ (octadecyl) groups. The coatings can be mechanicallyapplied by in situ crosslinking of polymers or can be functional groupscovalently bonded to the surface of the particles.

The amount of particles incorporated into the porous matrix can varywidely within the scope of the present invention. Generally, the amountof particles ranges from about 1 to about 99% by volume of the device.Preferably, the amount is greater than 20% by volume, and morepreferably greater than 50% by volume. Thus, a device of the presentinvention can contain up to 95% or more by weight of particles, therebyproviding a potentially high capacity for target attachment. Theparticles of the invention generally withstand a wide range of pHvalues, for example pH values about 4 or lower to pH values of about 12or higher.

The particles of the invention are versatile and are used to carry out avariety of chromatographic or non-chromatographic separation assays.Examples of the separation methods contemplated within the scope of thepresent invention include reverse phase separations, affinityseparations, expanded bed separations, ion-exchange chromatography, gelfiltration, chromatographic component separation, solid-phaseextraction, among other methods of separating, measuring or collectingchemical or biological target agents from other components of a sample.The particles are also used for binding to and thereby separatingnucleic acid molecules and/or polypeptide target agents from a sample.

A preferred particle of the device of the invention is a porous resin.Porous resins for adsorption separations are available in a largevariety of different materials, including silica, glass, cellulose,agarose, and a wide variety of different polymers, including polystyrenepolymethylmethacrylate, polyacrylamide, agarose, hydrogel, acrylicresins and other types of gels used for electrophoresis. Many of theporous adsorption resins such as silica, glass and polymers can be driedand have interconnected pores with surface areas in the range of about1-2 m²/g of dried resin to over 300 m²/g of dried resin. Other types ofresins are cross linked gels that cannot be dried without damaging thestructure. These types of resins normally do not have a specific surfacearea since the materials are able to diffuse uniformly through the crosslinked matrix.

Also encompassed within the scope of the invention is the use ofmodified resins including analogues, variants and functional derivativesof a natural or modified resin, or the functional groups thereof. Themodification includes for example, substitution, deletion, or additionof chemical entities (e.g., amino acids) to a particular resin, or itsfunctional group, or both. For example, amino substitution, acetylation,and/or partial acetylation of resins are included within the scope ofthe invention. Any modification to the functional group of a resin isalso included within the scope of the modified resins according to theinvention.

Other types of natural or modified resins useful within the scope of theinvention include, but are not limited to, phenyl sepharose, butylsepharose, octyl sepharose, polystyrene cross-linked with divinylbenzene, hydrocell C3 polystyrene-divinylbenzene, hydrocell C4polystyrene-divinylbenzene, hydrocell phenyl polystyrene-divinylbenzene,methyl HIC methacrylate,-Butyl HIC methacrylate, wide-pore-hi-phenyl,fractogel EMD, hydrophobic resin-propyl methacrylate co-polymerfractogel EMD, hydrophobic resin-phenyl methacrylate co-polymer octylsepharose, phenyl sepharose, Toyopearl HIC, Toyopearl amino-650S,Toyopearl amino-650M, Toyopearl amino-650C, Toyopearl amino-650EC,Toyopearl butyl-650S, Toyopearl butyl-650C, Toyopearl butyl-650M,Toyopearl ether-650S, Toyopearl ether-650C, Toyopearl ether-650M,Toyopearl hexyl-650S, Toyopearl hexyl-650C, Toyopearl hexyl-650M,Toyopearl phenyl-650S, Toyopearl phenyl-650C (PRDT), Toyopearlphenyl-650M, and Toyopearl 659 CU (PRDT) among others. All Toyopearlresins are available commercially from Tosoh Biosep, Montgomeryville,Pa. Sepharose resins are available from GE Healthcare, Piscataway, N.J.Fractogel resins are available from Merck, Darmstadt, Germany. Hydrocellresins are available through BioChrom Labs, Inc., Terre Haute, Ind. Theremaining resins are generic names for a variety of base materials forresins that are publicly available.

If porous resins are packed into a column, the hydrodynamic diameteravailable for flow is determined by the particle diameter and the bedvoid fraction:

$\begin{matrix}{D_{h} = {\frac{d_{p}}{3}\frac{ɛ}{1 - ɛ}}} & (1)\end{matrix}$wherein D_(h) is the equivalent hydraulic diameter for flow betweenparticles, dp is the particle diameter, and ε is the void fraction

As a result, to allow large species to flow through the column, it isnecessary to use large particles that in turn increase the diffusionresistance for adsorption into the resin. For example, to allow redblood cells to flow through the column, around 65 μm diameter particlesare necessary to provide 14 μm pore diameter in the interparticle spaceif the bed porosity is about 0.4.

4. Resin Impregnated Nonwoven Fabrics (RINs)

The incorporation of the particles into the matrix can be accomplishedthrough variety of ways. Since nonwoven fabrics can be made with acontrolled mean pore diameter, it is possible to impregnate porous resinparticles such as the ones described above, within the fibers making upthe nonwoven fabrics.

These impregnated nonwoven fabrics can be made in a variety of ways. Forexample, dry particles can be hydroentangled between two previouslyformed nonwoven fabrics. Alternatively, dry particles can be introducedwhile the fibers are being formed during meltblowing or spunbonding. Itis also possible to entangle resin particles while wet using wet layingprocesses. In one embodiment, the particles are impregnated into analready formed fibers by hydroentanglement and there would be no meltbonding of the particles with the polymer fiber matrix.

One preferred fabrication method is the direct calendering of alreadyprepared nonwoven fabrics that could be either meltblown or spunbonded.In one embodiment, the nonwoven fabrics is spread uniformly withparticles that are delivered at a fixed mass rate by direct calendaringso that the membrane is covered with a given mass of particles per unitarea. Once the particles are spread, a second membrane is placed overthe first to make a sandwich and the combination is passed through acalendering roll with a pattern that is able to bond the two membranestogether at low temperature and pressure. Particle densities on thesurface are in the range of about 0.1 to about 10 gm/m2 or more. Thepore size of the membranes used for this device allows larger entitiessuch as, for example, red blood cells to pass through since their poresize is larger than 10 μm. The particles in the membrane are attached toa ligand that facilitates binding of the particles to target agents suchas, for example, prion proteins from red blood cell concentrate andplasma.

The operation of the calendering process usually requires a hightemperature for the bonding of the nonwovens, but the temperature iskept below the melting temperature of the particles and does not affecttheir performance. In one embodiment, larger or denser particles mightbe placed between the nonwoven membranes by hydroentanglement.

The density and weight of the nonwoven fabric can take on a wide rangeof values to ensure a high particle density on the fabric whilemaintaining the desired pore dimensions. Particle concentrations ofapproximately 60% w/w can be impregnated into the fabrics. All commonmethods of making nonwoven fabrics can be used for this procedure,including fabrics with two different polymer fibers as well asco-extruded fibers with two different polymers. Because of thisflexibility, both wet and dry resins can be impregnated. If necessary,chopped fibers can be embedded into the fabric to facilitate the captureof the particles while still allowing flow pores of the necessarydimensions.

Chopped fibers are usually less than ½ inch long, and they are preparedby cutting a single fiber that is wound around a spindle or roll. Thechopping is achieved mechanically using rotating blades or other sharpcutting surfaces. Chopped fibers can be made from a variety of polymericor carbon fiber having a range of diameters from very small (less thanabout 1 μm) to large (>100 μm). In one embodiment, specific ligands arechemically grafted or coated on the fiber and then the fiber is cut tolengths of approximately ½ inch. Chopped fibers can then be distributedover a single layer of nonwoven fabric (polypropylene or other polymer)with a pore size and fiber diameter suitable to allow large entitiessuch as red blood cells, to pass through the membrane (>10 μm poresizes).

Chopped fibers can be delivered to the membrane at a prescribed rate toensure uniformity in the distribution of fibers. Once chopped fibers areon the surface, a second layer of nonwoven fabric can be placed on topof the chopped fibers and the combination can be passed through acalender to bond the two nonwoven layers. In one embodiment, a porousmatrix membrane or chopped fibers are functionalized with a ligand andis placed within another membrane, by for example air lay technology. Inanother embodiment, the ligands are attached to a polymer that issubsequently extruded into a fiber. The fiber can be chopped to makesmall segments that could be readily integrated between two membranes.

5. Surface Modification

Also included within the scope of the present invention are surfacemodified nonwoven or woven fabrics (SMNs) and surface modified particlesthat are functionalized on one or more internal and/or external surfaceswith a reactive group. Functionalization is achieved by addition of oneor more reactive groups to a surface of the porous matrix (e.g., wovenor nonwoven fabrics), particles or both. The reactive group interactswith and binds the target agent. The interaction between the reactivegroup and the target agent is a chemical, physical and/or a biologicalinteraction.

In one embodiment, the porous matrix, the particles or both are surfacemodified with a functional group capable of forming a covalent chemicalbond with a target agent. Functional groups useful within the scope ofthe invention include, but are not limited to, one or more of thefollowing groups, epoxy, formyl, tresyl, hydroxysuccinimide esters,among others. Other groups useful within the scope of the inventioninclude, but are not limited to, one or more of the following groups,sulfonic acid, quaternary amines, carboxylic groups, primary amines,cyano, cyclohexyl, octyl, and octadecyl groups, oxirane,N-hydroxysuccinimide esters, sulfonyl esters, imidazolyl carbamate,quaternary amines, carboxylic groups, dye ligand, affinity ligand,antigen-antibody, nucleic acid molecules, groups for ion exchange,chelation, oxidation/reduction reactions, steric exclusion reactions,catalysis reactions, hydrophobic reactions, reverse phase, and otherreactions normally encountered in chromatographic separations.

The functional group, for example ligands, are chemically conjugated tothe support or can be attached via linkers, such as streptavidin, betaalanine, glycine, polymers containing glycine-serine, short chainhydrocarbons of the formula —(CH₂)—, polyethylene glycol, epsilon aminocaproic acid, and linkers comprising —O(CH₂)n, wherein n is 1-30. Ifdesired, the ligand(s) can be attached by one or by several differentcleavable linkers, e.g., photolabile or acid labile moieties, enablingthe selective detachment of a population of ligands for analysis.Detached ligands can be used, for example, as affinity purificationmedia for proteins and enantiomeric separation (e.g., to concentrate,isolate, detect, characterize, quantify, or identify targets in asample), as diagnostic therapeutic tools, catalysts and enhancers ofchemical reactions, and as selective stabilizers of proteins.

In one embodiment, nonwoven membranes are coated with affinity ligandsas a functional group, which affinity ligands have specific affinity forprions on their surface. The example of affinity ligands includes, aprimary amine with a hydrophilic spacer containing polyethylene glycolunits. The ligands can be placed on the membrane (e.g., plasma treatedpolypropylene from Macopharma) through chemical grafting or by a latexemulsion coating method (padding).

5.1. Polymerization of Ligands on Porous Matrix

Polymerization of monomers on a porous matrix introduces epoxy groups onthe surface of these matrices, which in turn facilitates chemicalattachment of the ligand to the surface of the matrix. In oneembodiment, a monomer emulsion is applied onto cotton, polypropylene,polyester, and nylon fabrics by padding. Padding is a continuous processthat is used in the textile industry for dying, bleaching, and coatingof fabric. Additional information on padding is found in the CelaneseLLC web site: www.vectranfiber.com, incorporated herein by reference.Padding in general consists of a set of squeeze rollers used toimpregnate a fabric with a liquid by continuous passage of fabricthrough the liquid and then between the rollers to squeeze out excesssolution. It is possible to use a single-dip, single-nip paddingtechnique. Habeish et al., IMPROVING COTTON DYEING AND OTHER PROPERTIESBY EMULSION POLYMERIZATION WITH GLYCIDYL METHACRYLATE, American DyestuffReporter, April, 26-34 (1986), incorporated herein by reference,) haveapplied glycidyl methacrylate (GMA) emulsions to cotton fibers usingpadding techniques. After padding, the excess water is evaporated andthe polymerization is carried out at elevated temperatures. The amountof polymer on the fiber surface is in the range of from about 1 to about10% or more. The polymerization can also be carried out on nonwoven websof PET, PP, etc. with the desired pore size (>10 μm).

5.2. Latex Coatings on Fabrics

Latex emulsions are synthesized by convention emulsion polymerization inwater to make small particles of the desired polymer. Various solubleand free radical initiators and non-anionic and anionic surfactants canbe used to create the emulsions. In one embodiment, the latex emulsionsis coated on the porous matrix by padding as described above on eithersingle fibers or nonwoven webs of PP, PET, and other polymers. Anexample of this type of approach is provided by De Boos and Jedlinek,APPLICATION OF EPOXY FUNCTIONAL POLYACRYLATE EMULSION TO TEXTILES, J.Macromol. Sci-Chem. A17(2), 311-235 (1982), incorporated herein byreference.

6. Methods of Use

The methods, kits and devices of the invention are useful in a varietyof applications including prognostic, diagnostic, detection,purification, separation, processing of expressed in vitro geneproducts, and production and delivery of biopharmaceuticals. Thisinvention is applicable to any device that is commonly available formembrane operations, from flat plate, spiral wound or even hollow fibercartridge devices. Flow can be induced through the device by any commonmeans, from gravity to pumps, depending on the pressure drop and flowrate desired.

The purification and extraction techniques of the invention offeradvantages over conventional purification techniques, by reducing thenumber of purification steps, improving yields, increasing purity, andovercoming limitations associated with the traditional methods.

The devices of the invention are highly sensitive capable of separatingminute amounts of pathogens from a sample. In one embodiment, thedevices of the present invention are used for the removal of pathogenssuch as prions incluing PrP^(c), PrP^(sc), PrP^(res), viruses, bacteriaand toxins from whole blood, red blood cell concentrates, plateletsconcentrates, plasma, plasma derivatives, leukocytes, leukodepletedblood, mammalian cell culture, fermentation broths and other media usedfor the production and delivery of biopharmaceuticals and thepreparation of therapeutics. Multiple pathogens can be separated fromthe sample concomitantly and rapidly by the devices of the presentinvention from any stream in the plasma processing industry aimed at theproduction of therapeutic and/or pharmaceutical products.

In particular, the methods and devices of the present invention optimizethe protein purification process and improve the manufacturing processof biopharmaceuticals by increasing efficiency and purity.Biopharmaceuticals are drugs that are proteins, peptides or othercomplex polynucleotides or protein based macromolecules (collectively“gene products”). Their manufacturing process involves the recovery ofthe desired gene product from its host biomass, such as plasma or otherhuman and non-human biological sources (e.g., recombinant ornon-recombinant cell cultures, milk of transgenic animals or recombinantor non-recombinant plant extracts). Separating commercially viableyields of the desired protein from a biomass is challenging since thelatter contains unwanted host proteins, nucleic acid molecules and othernaturally occurring chemical entities.

Protein separation and purification processes present unique challengesdue to the variety of proteins, the different nature of possiblecontaminants and impurities, and the quantity of product to separatefrom the media. Conventional purification technologies generally involvea series of purification steps. With each step, the yield decreases andmanufacturing costs increase. Protein separation and purification coststypically represent over 50% of the total manufacturing costs.

In another embodiment, the devices of the invention are designed so thatthey perform two simultaneous operations: filtration as well asadsorption. In this embodiment, the fabric pore size is reducedsufficiently to reject large particulate material while at the same timemaintaining a pore size large enough to allow passage of the samplecontaining the desired molecule. This technology achieves simultaneousfiltration and adsorption steps in a single device and replaces membranefiltration followed by adsorptive column chromatography. For example,the devices of the invention make it possible to adsorb a desired or anundesired molecule secreted extracellularly directly from a culturemedium.

In another embodiment, the devices of the invention are used as analternative to columns for adsorptive removal techniques in thebiotechnology industry. These techniques utilize biochemicalinteractions such as, for example, ion exchange, chelation,oxidation/reduction reactions, stearic exclusion, catalysis, hydrophobicinteraction, reverse phase, dye ligand, affinity ligand,antigen-antibody and other interactions normally encountered inchromatographic and/or other separation techniques.

7. Test Kits

Also encompassed within the scope of the invention are test kits forsample purification by separation of target agents from the sample.

Complete test kits contain solutions and devices for target separationand purification of biological samples. For example, the test kitcontains a 96, 384, or 1536 well plate for high throughput samplepurification, and/or solutions for attachment of ligands to theparticles within the device of the invention in order to customizeindividual proteins, antibodies, and solutions required for proteinseparations in a plate format.

Generally, the test kits of the invention contain one or more of thefollowing: (1) one or more containers containing the devices asdescribed herein; (2) instructions for practicing the methods describedherein; (3) one or more assay component; and (4) packaging materials.The devices described herein are packaged to include many if not all ofthe necessary components for performing the separation methods of theinvention. For example, test kits include the device containing theporous matrix and particles in addition to one or more of the, buffers,reagents, chemical agents, functionalization reagents, enzymes,detection agents, control materials, or the like, among others.

In one embodiment, the kit additionally contains the functional groupsin separate containers, and the functional groups would have to beattached to the particles and/or porous matrix prior to performing anassay. Alternatively, the device may be provided in the kit withoutfunctional groups, in which case the porous matrix, particles, or bothare preferably pre-functionalized.

The devices of the invention can be of any desired size and shape.Preferably the device is a sheet-like material which, for example, is ina disk or strip form. Other items which may be provided as part of thetest kit include solid surface syringes, pipettes, cuvettes, andcontainers. Coating the porous matrix or particles with monolayermaterials or thicker materials provided by in-situ cross linking ofpolymers or covalently bonding functional molecules on the surfaces ofthe porous matrix or particles allows the optimization of bothchromatographic selectivity and separation efficiency.

Detection can be facilitated by coupling the porous matrix, particles,or both to a detectable agent. Examples of detectable agents include,but are not limited, to various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, radioactivematerials, disperse dyes, gold particles, or a combination thereof.

EXAMPLES

It will be understood by one of ordinary skill in the relevant arts thatother suitable modifications and adaptations to the methods andapplications described herein are readily apparent from the descriptionof the invention contained herein in view of information known to theordinarily skilled artisan, and may be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

Example 1 Surface Modified Nonwoven Fabrics (SMNs)

Surface modified nonwoven fabrics are specifically useful when thetarget species to be adsorbed is large, and it is unable to penetratethe pores of the resins. In this instance, the surface of the fiberscomprising the nonwoven fabric was modified to affect the adsorption ofthe target agents. The adsorption step involves ion exchange,hydrophobic, or affinity interactions or any other common adsorptionprocesses. If SMN is used without the particles, the surface area perunit volume of material available for attachment is controlled by theporosity of the fabric and the diameter of the fibers,

$\begin{matrix}{a_{v} = {\frac{4}{d_{f}}\left( {1 - ɛ} \right)}} & (2)\end{matrix}$Wherein a_(v) is the specific surface area per unit volume of solid,d_(f) is the fiber diameter, and ε is the void fraction.

Since fiber diameters anywhere in the range of 100 nm to 10 μm areavailable, and porosities normally are in the range of 0.4-0.5, verylarge surface areas can be achieved in these devices. For example, witha fiber diameter of 0.1 μm, the surface area per unit volume of fabricwould be on the order of,a _(v)=2×10⁷ m²/m³=20 m²/cm³  (3)

This compares quite favorably with the surface area per unit volume ofmany porous chromatographic supports. However, since the mean pore flowdiameter of the fabric can be controlled independently, the pore sizescan reach several microns in diameter. Techniques such aselectrospinning are able to produce even smaller diameters, resulting inmuch larger areas per volume.

Any surface modification that facilitates binding of a target agent tothe device and is compatible with the chemistry of the specific porousmatrices used in the device is encompassed within the scope of theinvention. Surface modification includes, for example, the formation ofcharged species, the attachment of affinity ligands, peptides,oligonucleotides, proteins, spacer arms, hydrophobic moieties,fluorinated materials, among others.

Since the surface of the fibers in nonwoven fabrics tend to be smooth,these surfaces present a preferred configuration for the exposure ofaffinity ligands to a particular large species such as prion proteins, avirus or a bacterium.

Example 2 Device Configuration for Prion Protein (PrP) Removal

This example demonstrates the possibility of designing different deviceconfigurations to remove endogenous transmissible spongiformencephalopathy infectivity by allowing adsorption to non-porous surfacesof various geometries. Endogenous infectivity from red blood cellconcentrates involves the removal of infectious PrP^(sc) (scrapie formof the prion protein) or PrP^(res) (resistant form of the prion protein)at a total concentration of approximately 200 ng/ml. In a bag of redblood cell concentrate (rbcc) containing 350 ml, there was a total of7×10⁻⁵ g of PrP. Given that a monolayer of protein coats a surface witha monolayer of density of approximately 2 mg/m², the total surface arearequired for binding all of the endogenous PrP in rbcc is estimated asfollows.

$\begin{matrix}{A = {\frac{7 \times 10^{- 5}g}{2 \times 10^{- 3}{g/m^{2}}} = {3.5 \times 10^{- 2}m^{2}}}} & (4)\end{matrix}$Wherein A is the total area of the device.

As it is evident from the equation above, the total surface are requiredfor binding prions is relatively small and accommodates several devicegeometries suitable for exposing affinity ligands at the correct surfacedensity.

A. Square Sheets

A set of N square sheets of nonwoven fabric having both sides coatedwith ligands exposed to the blood has a total surface area given by,2NL ²=3.5×10⁻² m²  (5)Wherein N is the number of sheets, and L is the length/width of a squaresheet. In the case of 10 sheets (N=10), the required width of each sheetwould be,L=0.042 m=4.2 cm  (6)

A device of this type consists of a staggered array of sheets with thefluid flowing in the tortuous path between sheets, as demonstrated inFIG. 2.

B. Coated Fibers

A set of N nonwoven fibers coated with affinity ligands on the outsidehave a total surface are given by,N2πRL=3.5×10⁻² m²NRL=5.57×10⁻³ m²  (7)Wherein R is the radius of a fiber.

For example, the number of fibers of radius of 5 μm and length 5 cmwould be,N=22,280  (8)The volume of these fibers would be,

$\begin{matrix}{V_{f} = {{N\;\pi\; R^{2}L} = {{8.74 \times 10^{- 8}m^{3} \times \frac{10^{6}\mspace{14mu}{ml}}{m^{3}}} = {0.087\mspace{14mu}{ml}}}}} & (9)\end{matrix}$Wherein V_(f) is the volume of fibers.

As it is evident from the equation above, the volume of the fibers wererelatively small, which is primarily due to the very small fiberdiameter that gave rise to a very high surface area per unit volume. Inorder to allow proper flow of red blood cells through such a fiber mat,the porosity would have to be fairly high, for example about 50%. Inthis case, the volume of the device would be roughly twice the fibervolume or 0.17 ml. This is a very small volume, again indicating that adevice for this type of capture does not need to be large to meet thecapacity requirements. For example, a fiber mat of 2 cm radius wouldonly have to be approximately 0.135 mm thick to provide this volume. Oneor more sheets of fibers can be coated with affinity ligands on theoutside.

C. Particles

Small non-porous particles also exhibit a very high surface area perunit volume.

The number and volume of the particles required to have the surface areastated above was computed in a manner similar to that used in the caseof cylindrical structures,

$\begin{matrix}{{N = \frac{3.5 \times 10^{- 2}m^{2}}{4\;\pi\; R^{2}}}{V_{s} = {\frac{4}{3}\pi\; R^{3}N}}} & (10)\end{matrix}$Wherein, Vs is the volume of a particle.

Given particles of radius 10 μm, the numbers and volume of particlesgiven by equation 5 are,N=2.79×10⁷V _(s)=1.17×10⁻⁷ m³=0.117 ml  (11)

The equation shows that an extremely small volume of small particles.Small particles could be dispersed with larger particles or suspended ina cross-linked gel (such as agarose) with large pores to allow easy flowof red blood cells through the system.

Example 3 Bonding Two Layers of Membranes by Calendering With or WithoutResin

In order to develop a prion removal device, two layers of polypropylenemembranes were calendered successfully under room temperature/400 PLIand 150F/100 PLI, with resin density at 1 mg/cm². Calendered membraneswere sealed using an ultrasound sealer. The percentage of hemolysis fromcalendered membrane samples was well within the acceptable limit.Calendering was used for impregnation of Amino 650M resin between twomembrane layers.

Toyopearl Amino 650M resin particles were impregnated between two layersof nonwoven fabric membranes. Polypropylene (Inner layer) and polyester(Outer layer) membranes that are currently used in MacoPharmaleucofilters were good candidates for these membranes since they havealready been approved for processing human blood.

In order to investigate whether particles could be immobilized withouthindering the flow of red blood cells through the device, the innerand/or outer layer membranes were calendered with or without particles.Calendering was achieved by pressing membranes between two rollers intosheets.

Materials And Methods

One roll of polypropylene membrane (PP) and one roll of polyestermembrane (PET) were wound on a 3-inch internal diameter plastic spindle.The width of the rolls was about 0.5 meters. The membranes were 22.5 cmwide and 800 m long. Membranes were cut into 22.5 cm×22.5 cm squaresheets. Dry resin was spread at 1 mg/cm² on one side of membrane, andthen covered with another membrane. Membrane alone samples did not needthe resin spread. The above samples were passed through two calenderrolls, one embossed roll and one smooth roll. Both rolls can be heatedto increase temperature for calendering. The pressure between the rollswas also controlled. Calendered samples were tested by visualexamination, weight measurement, cross-section examination by SEM(scanning electron microscopy) pore size test, and percent hemolysistest of flow through after passing whole blood through calendereddevice.

Procedure for Measurement of Percent Hemolysis

Membrane samples were cut into 25 mm circles, and placed into MilliporeSwinnex 25 mm filter holders. Each sample was tested in duplicate onflow through, and then triplicate on 96-well plates. Each sample wasrinsed with 2 ml working buffer (working buffer is 20 mM citrate and 140mM NaCl, pH 7.0), then whole blood was pumped through membranes from thetop at 0.5 mL/min. Five ml of flow through were collected from eachsample. Flow through or untreated blood was centrifuged at 12000 rpm for10 min at 4° C. to take the supernatant. Three 100 μL aliquots from eachsample were placed into three wells of a 96-well plate. The UVabsorbance of each plate was read at 415 nm. The average value ofA_(415 nm) was divided by the value from 100% lysis of the same blood.The percentage of hemolysis is acceptable if it's below 1%.

Results

TABLE 1 Conditions used for calendering and visual examination Samplesmade by calendering: Temperature (F.) Sample Membranes Embossed SmoothPressure No. calendered Resin roll roll (PLI)* Results 1 Inner/Innerlayer Yes cold cold 100 with big area of pocket 2 Inner/Inner layer Yescold cold 200 with medium area of pocket 3 Inner/Inner layer Yes coldcold 300 with small area of pocket 4 Inner/Inner layer Yes cold cold 400with isolated small area of pocket 5 Inner/Outer layer Yes cold cold 300barely bonded, big pockets 6 Inner/Inner layer Yes 120 120 100 withsmall area of pocket 7 Inner/Inner layer Yes 140 140 100 with isolatedsmall area of pocket 8 Inner/Outer layer Yes 150 150 100 not bondedwell, big pockets 9 Inner/Outer layer Yes 150 150 200 poorly bonded,some pockets 10 Inner/Outer layer Yes 180 180 200 poorly bonded, somepockets 11 Inner/Outer layer Yes 180 180 400 loosely bonded 12Inner/Outer layer Yes cold cold 400 barely bonded 13 Inner/Inner layerYes 150 150 100 very good, with little pocket 14 Inner/Inner layer Nonecold cold 100 loosely boned, with pockets 15 Inner/Inner layer None coldcold 400 tightly bonded without pockets 16 Inner/Inner layer None 157153 100 very well bonded 17 Inner/Inner layer None 157 153 400 tightlybonded 18 Inner/Outer layer None 157 153 400 loosely bonded 19Outer/Outer layer None 180 180 400 barely bonded, big pockets 20Outer/Outer layer None 220 220 400 bonded with some pockets 21Outer/Outer layer None 220 220 600 very well bonded *PLI = pounds perlinear inch

From visual inspection, sample Nos. 4, 7, and 13 determined to be thebest ones for particle embedment. When increasing the temperature of therolls, a lower pressure can be used to bond the membranes as shown forsamples 4 and 13. The outer layer membrane was made of polyester thatwas much thicker and stiffer than the inner layer. To calender the outerlayer with either outer or inner layer membrane, higher rolltemperatures and pressures were required as shown for sample 21.

TABLE 2 Weight measurement Weight of calendered samples: Samples ResinWeight (gsm) Single inner layer 41 Single outer layer 67 Inner/Innerlayer calendered 82 Inner/Inner layer calendered 1 mg/cm2 92 Inner/Outerlayer calendered 107 Inner/Outer layer calendered 1 mg/cm2 117Outer/Outer layer calendered 1 mg/cm2 129

For resin density, 1 mg/cm² equals to 10 g/m² (1 mg/cm²×10⁴ cm²/m²=10g/cm2). The weight from single layer, double layer and double layerembedded with resin was relatively proportional. The results showed thatresin was well maintained between two layers of membranes.

The scanning electron micrograph (SEM) of samples 11 and 13 revealedthat most resin particles were intact after calendaring, even thoughsome were cracked. Sample 2 was also examined by SEM and revealedsimilar results. On the embossed roll of the calender, there were squaregrid spaces 2 mm×2 mm. During calendaring, the membranes were highlypressed where the grids touched. This area is referred to as the bondingarea. The pocket area refers to an area that is farthest from thebonding area.

Sample 11 is an example of immobilizing resin particles between oneinner layer and one outer layer. FIG. 3 shows pocket areas of sample 11at 50× magnification. Sample 13 is an example of immobilizing resinparticles between two inner layers. FIG. 4 shows pocket areas of sample13 at 50× magnification.

Pore Size Distribution

The results of the pore size distribution of calendered samples areshown in Table 3 below. For calendered samples, the smallest, mean, andlargest pore sizes decreased 30% to 50% compared with the single layer.In order to determine whether the decrease in the pore size would hinderthe passage of red blood cells through the device, further tests on thehemolysis of whole blood flow through were performed.

TABLE 3 Pore size distribution of calendered samples: Rolls TemperatureSample (° F.) Pressure Pore Size Distribution (μm) No. Membrane ResinEmbossed Smooth (PLI) Smallest Mean Largest  1 Inner/Inner layer YesCold Cold 100 2.28 3.62 7.71  2 Inner/Inner layer Yes Cold Cold 200 2.104.52 8.80  3 Inner/Inner layer Yes Cold Cold 300 1.91 3.89 7.80  4Inner/Inner layer Yes Cold Cold 400 1.99 4.69 9.62 15 Inner/Inner layerNo Cold Cold 400 2.06 3.90 9.26  7 Inner/Inner layer Yes 140 140 1001.82 4.05 8.00 13 Inner/Inner layer Yes 150 150 100 1.99 4.88 8.73 16Inner/Inner layer No 157 153 100 2.11 4.04 8.79 PP Single inner layer4.15 7.03 13.91 11 Inner/Outer layer Yes 180 180 400 1.12 3.32 7.99 18Inner/Outer layer No 157 153 400 1.64 3.45 8.65 21 Outer/Outer layer No220 220 600 N/A 17.76 47.35 PET Single outer layer 23.61 35.81 79.04

Example 4 Optimization of Calendering Using High Particle Densities

The calender roll used in this trial was ordered by ProMetic from BFPerkins. The roll is made of stainless steel, engraved with a honeycombpattern, and coated with Teflon release coating. The back roll used wasrubber coated (¾″ to 1″ thick).

Four-gram samples of dry resin were spread manually into 30 cm×20 cm(600 cm²) swatches of plasma-treated polypropylene membrane, whichcorresponds to a particle density of 6.6 mg resin/cm². A second swatchof membrane was placed on top of the resin layer, and the sandwich waspassed through the calender rolls at 10 m/min. The following tablecontains the results obtained during the tests.

TABLE 4 Calendering optimization results Set Measured temperaturetemperature Gap¹ Trial (° F.) (° F.) (μm) Results 1 212 198 203 Nobinding with or without particles 2 230 215 0 Membranes were weaklyfused 3 245 236 0 Better than previous, but still too weak 4 255 245 0Good binding without resin, but less efficient with resin 5 260 248 0Good results with and without resin 6 275 — 0 Temperature was too high,top membrane did not fuse to bottom, but adhered to the roll 7 265 — 0Good results were achieved at this temperature with and without resin ¹Azero gap indicates that the pattern penetrates the bottom roll by a1/1000 of an inch.

The samples were observed under the microscope, and showed no pinholes.A swatch of each trial was kept (only without resin) for futurereference.

Example 5 Binding of a β-Lactoglobulin and Flow Characteristics ofResin-Impregnated Calendered Membranes

This experiment was conducted to determine the breakthrough curves forthe binding of a model protein (β-lactoglobulin) to calendered membranematerials containing dry resin at a density of 4 mg/cm².

Polypropylene membrane material was calendered at 170° F. and 150 poundsper linear inch (PLI) containing dry resin at a density of 4 mg/cm².This membrane material was cut and assembled into Millipore Swinnexfilter units. Each filter unit contained a stack of 1 to 4 membranelayers plus a layer of non-calendered membrane at the exit side of thefilter unit. A solution of 0.5 mg/mL β-lactoglobulin in 1× PBS waspassed through the filter unit at 1.5 mL/min using a peristaltic pump.Fractions of 0.5 mL were collected for 4 minutes and analyzed for theirprotein concentration using the Pierce Micro BCA assay kit (Pierce,Rockford, Ill.).

FIGS. 5-7 show the results of the Micro BCA assay of the 12 fractionscollected for the different number of membrane layers. Calenderedmembrane without resin was used as the control. The results from thisexperiment show the difference in binding between the membrane withentrapped resin and the control. All of the runs displayed a similarinitial slope of unbound concentration; however, layers 2-4 were not runlong enough to show the saturated condition.

TABLE 5 Total bound protein and the amount of protein bound per weightof resin. Total Protein Bound mg Membrane Control Resin mg bound/g resin1 Layer 0.805 1.095 4.5 2 Layers 0.792 1.493 5.4 3 Layers 0.731 2.0856.9 4 Layers 0.776 1.859 4.2

The amount of protein bound in general increased for each additionallayer of membrane peaking at three layers of membrane followed by a verysmall decrease with four layers (Table 5). Since the filters containing2-4 layers of membrane were not run long enough to display saturatedconditions it is not certain that they were done binding.

Example 6 Particle Distribution on Membrane Rolls

A particle spreading unit was developed to replace the manualdistribution of beads used previously. The equipment has been tested andcalibrated.

The powder applicator was manually set for 60%, which was equivalent toa dispensing rate of 6.52 to 6.99 Kg/hour (displayed). The measureddispensing rate determined by weight was 6.65 Kg/h (average of threedeterminations), within 5% of the target value of 6.96 Kg/hour.

The powder dispensed appeared to be distributed with uniformity (visualevaluation) and no spilling over the edges of the membrane. FIG. 9 showsthe distribution of particles onto the bottom membrane after running theline for about 1 hour. The sharp edges formed by the area containing theparticles can be noticed on both sides of the membrane. Anothernoticeable feature is the lack of powder on the conveyor belt, evenafter some production time.

Example 7 Prions Binders

A list of resins used for binding prions is disclosed below.

a) Amino 650M—Base resin for coupling of peptide and other ligands. Thisbase resin has demonstrated utility in binding of prion protein, bothnormal PrPc and infectious PrPsc (or PrPres). The resin was used incolumn chromatographic format and we have demonstrated removal/bindingof PrPsc (hamster, mouse vCJD, mouse Fukuoka, human spCJD and HumanvCJD) from red blood cell concentrate, plasma, whole blood to the limitof detection by in-vitro techniques (Western Blot) and a reduction inhamster 263K scrapie infectivity, i.e., in-vivo model (red blood cellconcentrate) of approx. 4 logs.

b) Toyopearl—SYA—This tripeptide has demonstrated utility in binding ofprion protein, both normal PrPc and infectious PrPsc (or PrPres). Theresin was used in column chromatographic format and we have demonstratedremoval/binding of PrPsc (hamster, mouse vCJD, mouse Fukuoka, humanspCJD and Human vCJD) from red blood cell concentrate to the limit ofdetection by in-vitro techniques (Western Blot) and a reduction inhamster 263K scrapie infectivity, i.e., in-vivo model of approx. 4 logs.

c) Toyopearl—DVR—This tripeptide has demonstrated utility in binding ofprion protein, both normal PrPc and infectious PrPsc (or PrPres). Theresin was used in column chromatographic format and we have demonstratedremoval/binding of PrPsc (hamster, mouse vCJD, mouse Fukuoka, humanspCJD and Human vCJD) from red blood cell concentrate to the limit ofdetection by in-vitro techniques (Western Blot) and a reduction inhamster 263K scrapie infectivity, i.e., in-vivo model of approx. 4 logs.

The amino 650M, SYA and DVR have been used at full scale, i.e., 1 fullunit of red blood cell concentrate passed over the resin (approx 350ml). Column size was 10 ml of swollen resin. SYA, DVR, and aminofunction at 400 μmol/g (dry resin).

Example 8 Comparison of PrPsc Binding to Amino 650M and Amino 650U fromSBH Spiked into Buffer, Filtered Plasma, and Whole Blood

Amino 650U is a mixture of different bead sizes that includes Amino 650Mand it is less expensive to produce than 650M. Amino 650U was tested forendogenous PrP and for its ability to bind PrP^(sc) in all the matricescurrently used, buffer, filtered plasma and whole blood and it wascompared to binding with Amino 650M challenged with spiked whole blood.The experiment was designed to compare the binding of PrP^(sc) fromspiked buffer, plasma, and whole blood to Amino 650U and to establishbinding of endogenous PrP^(c) from plasma and whole blood to Amino 650U.Additionally, the experiment was designed to determine the effect ofleukofiltration in the removal of PrP^(c). Spiked buffer refers to theaddition of brain homogenate to working buffer. Spiked whole blood isthe addition of brain homogenate to human or hamster whole blood.

No difference in the signal was found for prion removal by 650 U or 650M when present in plasma or whole blood. In conclusion amino 650 Uand650 M performed the same. The amount of PrP^(c) removed byleukofiltration was more than that estimated to be in platelets andleukocytes together. Thus, it was possible that leukofiltration capturedalso some of the plasma-derived PrP^(c). It has been shown thatleukofilters behaved differently with regard to capture of human andhamster plasma-derived PrP^(c). It is possible that while hamster plasmaPrP^(c) was not captured by the filter, human plasma PrP^(c) was.Finally, it is also likely that the difference between the two resultsis due to lack of correlation between PrP^(c) and infectivity.

The amount of PrP^(c) removed by leukofiltration was more than thatestimated to be in platelets and leukocytes together. Thus, it waspossible that leukofiltration captured also some of the plasma-derivedPrP^(c). It has been shown that leukofilters behaved differently withregard to capture of human and hamster plasma-derived PrP^(c). It ispossible that while hamster plasma PrP^(c) was not captured by thefilter, human plasma PrP^(c) was. Finally, it is also likely that thedifference between the two results is due to lack of correlation betweenPrP^(c) and infectivity.

Example 9 Binding of Hamster Brain PrP^(sc) to AMN Resins

Comparative binding experiments were conducted for a series of resins(e.g., AMN-13, 14, 15, 16, and 17, Amino 650M and Amino 650U). AMNseries relate to 650 U (newly designated as 650C-prdt) samples withvarying amino substitution levels as follows:

-   AMN-13; 0.094 eq/L-   AMN-14; 0.078 eq/L-   AMN-15; 0.072 eq/L-   AMN-16; 0.063 eq/L-   AMN-17; 0.098 eq/L

The resins bound to PrP^(sc) from spiked buffer, plasma, and wholeblood. The results demonstrated that all AMN resins bound equally wellwhen challenged with both spiked buffer and spiked whole blood.Furthermore, the signal with AMN resins was the same as that with amino650 M and 650 U. Comparing the resin binding of PrP from spiked plasma,there was a slightly more intense signal from Amino 650M compared to allother resins. Among the AMN resins #13 appeared to have weak PrP signal,but very comparable to amino 650 U while #15, 16, 17 all performedbetter than amino 650 U. No noticeable difference was observed betweenAMN 14, 15, 16, 17 resins.

In conclusion, the study demonstrated more similarity among the resinsand most importantly it showed closer correlation with amino 650U thanwith 650 M. The differences observed with plasma suggested that at leastwith that challenge reducing the level of substitution may be beneficialand the resin performed more closely to amino 650 M.

Example 10 Extraction of Proteins Bound to Resin-Embedded Membranes andDetermination of Binding of PrP^(c) from Normal Hamster Brain Homogenate

The development of the new device using resin-embedded calenderedmembranes lead to the need of developing new procedures for extractionof the bound proteins from the resins. Changes had to be made to thehandling of the material, as well as the composition, concentration andvolume of the extraction solution. The experiment was also designed toperform binding evaluations in the new format, using both ToyopearlAmino 650Mresin-embedded membranes and its fully acetylated form.

Normal hamster brain homogenate (HaBH) was treated with sarkosyl andspun down. The resulting supernatant was diluted to a finalconcentration of 1% using working buffer or human whole blood. Fiftymilliliters of spiked solution was passed through 47 mm Swinnex filterholders (Millipore) containing 4 sandwiches of calendered membranesembedded with 4 mg/cm² of chromatographic resin at full capacity, or areduced substitution capacity form of the same resin as a control eitherToyopearl Amino 650M or its fully acetylated form. The flow rate usedwas 0.5 mL/min, using a peristaltic pump. Ten aliquots of 5 mL each werecollected for each of the spiked solutions and membrane type. Theflowthrough samples of both membranes challenged with spiked buffer wereanalyzed by ELISA. The membranes containing resins fully acetylatedresin and challenged with spiked whole blood were rinsed using workingbuffer.

Sections of membranes (in some cases the whole stack) were treated witheither SDS-PAGE sample buffer or 99% formic acid. Treatment with formicacid consisted of adding 0.5 mL of 99% formic acid and 10 μL of 20% SDSto 1 quarter of a membrane sandwich, followed by incubation for 1 hourremoval of the liquid, and evaporation using a SpeedVac. The samples hadtheir volumes adjusted to 15 μL using water, followed by addition of 15μL of 2× sample buffer. The treatment with sample buffer consisted ofadding 3 mL of 1× sample buffer to the complete stack of membranes,followed by incubation for 30 minutes, and boiling for 7 minutes. Thesolution was harvested without pressing the membranes, and centrifugedbriefly to remove all the resin. A variation of the above treatment wasalso tested. It consisted of adding 1 mL of 2× sample buffer to twoseparate stacks of membranes corresponding to ¼ of a filter, incubatingfor 1 hour, followed by boiling only one of them. Elution with samplebuffer without boiling may be used if disassembling the filter holdersbecomes too risky when using infectivity.

A final condition tested was the incubation of sections (¼) of themembranes with sample buffer to verify binding to the first, second,third and fourth membrane to contact the challenge solution. Sampleswere then run on SDS-PAGE gels and stained for total protein. Westernblots were also performed. The void volume of the filter holder wasapproximately 7 mL. After passing 50 mL of challenge solution througheach of the filters, followed by air, the volumes recovered were 45 and47 mL for whole blood. When using spiked buffer, the volumes recoveredwere 46 and 46 mL. There was no significant difference noticed whenusing the different challenge solutions.

The first filter holder to be open was the one containing the membranewith fully acetylated Toyopearl that was challenged with spiked wholeblood. It was noticed that despite the passing of air and rinsing withbuffer there was still some blood inside the filter. During the attemptto rinse the membranes with buffer, there was a significant loss ofresin, and the membrane was discarded.

The filter holder with Toyopearl amino 650M challenged with whole bloodwas rinsed with an extra 200 mL of buffer. The flow rate was higher thanmax (999 in the dial). Upon opening the holder it was noticed that therewas still some blood inside, especially between layers. It was alsonoticed that a couple sections delineated by the radial distributor werebypassed during the wash.

The stack of membranes was cut into 4 quarters. One of the pieces hadthe four layers separated and treated with sample buffer to investigateif the different layers had different binding. Another quarter was alsoseparated into pieces and submitted to the formic acid treatment. Theremaining two quarters were used to compare the treatments with andwithout heating.

The two filters challenged with spiked buffer were rinsed with 200 mL ofworking buffer each. The filters were opened and the whole stack wastransferred to a small glass vial, to which 3 mL of sample buffer wasadded.

The resin embedded in the calendered membranes appeared to maintain thesame PrP binding properties characteristic of the resin in columnformat. The fully acetylated amino showed weaker membrane-bound PrPsignal compared to amino signal, supporting the conclusions that fullyacetylated amino may not bind PrP efficiently. In general, the resultsindicated that 50% accetylation whether in a blend form or by chemicalsynthesis reduced the PrP^(res) binding.

Example 11 Binding of PrP^(c) from Normal Hamster Brain Homogenate toFilters Containing Particle-Impregnated Membranes

The following experiment demonstrates the binding of normal PrP(PrP^(c)) from normal hamster brain homogenate (NBH) by membranescontaining resin particles.

The elution method described in the previous example was applied tothese samples. The filter was opened, the membranes were placed in aglass vial and incubated with mixing with 2 ml of NuPage sample buffer(Invitrogen corporation, Carlsbad, Calif.) The vial was then heated andthe resin that came out of the filter was collected. Western blotresults of the eluted proteins indicated that the method eluted PrP fromthe membrane. The results also indicates that the filters with theresins bound more PrP than the filter without the resin.

Example 12 Binding of PrP^(c) from Scrapie Brain Homogenate to FiltersContaining Particle-Impregnated Membranes

This experiment demonstrates the performance of filters in bindingPrP^(sc) from infectious hamster brain homogenate (SBH) spiked intowhole blood and in buffer. The filters contained membranes impregnatedwith full capacity resins, as well as reduced capacity resin, and noresin as control.

Elution was done with injecting 2 ml of NuPage sample buffer (accordingto extraction described in Example 10). Western blot of the elutedproteins showed strong signal without PK (protein kinase) but weaksignals with PK. Since the proteins were eluted with 2% SDS, the PKdigestion was conducted under 2% detergent concentration instead of 0.2%SDS (standard procedure). It is likely that the PrP^(res) weak signalwith PK is due to excess SDS in the reaction mixture. The resultsindicate weaker signal with the membranes without resin, but similarsignal intensities for all other resins tested. No difference wasobserved between SBH in buffer and in whole blood for full capacityresins and no resins. Reduced capacity resin showed stronger signal withbuffer spiked compared to blood spiked.

Example 13 Testing of Prototype Filter

In this example, the filters were assembled in plastic casings andwelded together, in a configuration similar to a final device. Theperformance of such device was evaluated using various challenges,spiked and non-spiked. In one embodiment, the final device was composedof a rigid or pliable plastic casing containing layers of non-calenderedmembrane (from 1 to about 25 or more), followed by several layers(between 1 and about 50, depending on the desired capacity of thedevice) of resin-embedded membranes, and between 1 to about 25 layers ofnon-calendered membranes. The filters were welded together using anultrasound cutter/sealer or by the use of a press to apply heat andpressure simultaneously. In this example, heat and pressure were used.

Hamster brain homogenate was treated with 0.5% Sarkosyl and diluted100-fold in buffer, filtered plasma, and whole blood and used as thechallenge to the resins. The resins were challenged with 80 ml of sampleat 0.5 ml/min with a peristaltic pump. Western blots of the resin boundPrP^(res) samples were conducted without PK digestion. The resultsindicated that the membranes performed well with the spike in buffer andin plasma but the same spike in blood appeared to be reduced.

Example 14 Removal of PrP from Spiked RBCC by a Series of Filter Devices

This experiment shows the removal of PrP from spiked red blood cellconcentrate (RBCC) using a device containing particle-impregnateddevice. Since the device was challenged with an excess of targetprotein, a series of devices was used. The total volume of RBCC used wasequivalent to one unit.

All filtrations went without any obvious problem and a pump was used forall filtrations. All filters had soft casings and were pre-tested forleakage. None of the filters leaked in the pre-test or in the actualexperiment. The filtration time was about 10 minutes each for one unitof RBCC (˜300 ml). Filters were washed with about 460 ml of workingbuffer (citrate). The efficiency of the washing step was empiricallyassessed by examining the color of the filter after washing.

After washing, the filters were injected with air to remove all liquidin the filter. The filters were treated with ˜4.6 ml of sample bufferfor elution (injected on one side of the filter). The sample buffer wascollected and injected on the other side of the filter. This step wasconducted three times. The results indicated that that PrP^(res) escapedfilter 1 and was captured by filter 2, which was expected, since thechallenge applied was higher than the capacity of one filter set.PrP^(res) could be also present in the eluate of filter 3 but be belowthe limit of detection of the Western blot. These results compared tothose with RBCC in the first PRDT infectivity study indicate that thefilters performed as well as the same resins used in a column format. Aswith the previous study, large excess of PrP^(res) was passed throughthe filters and it is not surprising that not all PrP^(res) was capturedin the first filter.

Example 15 Grafting Glycidylmethacrylate on Polypropylene, PolyethyleneTerephthalate, Cotton and Nylon Substrates

Grafting is an efficient method to change surface properties ofpolymers. The grafted polymers tailored with specific ligands incombination with superior mechanical properties of the substrates makegrafting the most versatile method for protein binding, whoseapplications often require change of ligands as well as enoughmechanical strength. In this example, grafting of glycidylmethacrylate(GMA) on polypropylene (PP), polyethylene terephthalate (PET), cottonand nylon substrates were performed to show the possibility of applyingthis approach for PrP^(c) removal from human blood.

Procedure:

The substrates used for the tests include cotton woven fabric, nylonwoven fabric, PP non-woven fabric, PP non-woven membrane (MacopharmaPP175), PP fibers and PET fibers from the College of Textile at NCSU.

Grafting was carried out by the following steps:

-   1. Samples were washed with acetone three times;-   2. Samples were dried in a vacuum oven for more than three hours;-   3. Samples were treated with Argon plasma for 15 s at 750 W;-   4. Samples were exposed to air for 30 minutes;-   5. Samples were then immersed in 10% GMA solution in a UV chamber    for 6 hrs. The UV intensity was 1.1 W/m². The temperature of chamber    was 30° C. and that of sample rack was above 60° C.;-   6. After the UV polymerization, samples were then washed with    acetone for three times;-   7. Samples were dried in a vacuum oven.

At this stage, samples were only analyzed for their weight gain and IRspectrum. After analysis, the grafted PP non-woven fabric and PPnon-woven membrane were further aminated in ammonia hydroxide solutionat 60° C. overnight. These samples were then washed and dried forelemental analysis.

Results:

From the results of gravimetric measurement and IR spectroscopy, the PPsubstrates show significantly better grafting results than the othersubstrates, cotton, nylon and PET. Three types of PP substrates havebeen tested, PP fiber, PP non-woven and PP non-woven membrane(Macopharma). The weight gains after grafting are 85% 154%, and 57%,respectively (Table 6). These values are much higher than those (−3% to4%) of the other substrates. These results are further confirmed by theIR spectra, where the grafted PP systems show strong peaks at 1720 cm⁻¹related to the carbonyl group and at 845 and 910 cm⁻¹ related to theepoxide group, the signature for GMA. Other substrates did not show muchchange when comparing the blank sample with the samples after grafting.

The reasons for the differences of grafting on the various substratesare still not clear. One possible reason is that C—H bond might beeasier to break in the PP environment than in the other environment.Another possible reason is that the nylon and cotton fabric samplesmight have had surface treatments unknown to us. Simply washing byacetone may not be sufficient to remove the finishing.

It is also interesting to note that for the original PP fiber andnon-woven membrane (Macopharma PP175) samples show peaks in the 840 to920 cm⁻¹ range. However, no such peaks are shown for the PP non-wovenfabrics. The peaks for the former could be the results of oxidation ofthe sample surfaces, which are from processing at high temperatures.

From the IR spectrum, the aminated Macopharma sample shows broader peakat 3400 cm⁻¹ which is attribute to —OH and —NH₂ groups, the results ofamination.

Determination of Surface Area of Non-Woven Materials

Determining surface area of non-woven materials is not easy task due tocomplex interlocks of fibers that forming the non-woven. However,surface area of a single fiber can be determined accurately by measuringmicroscopic images of the fiber and the length. Therefore, it istheoretically possible to measure surface area of non-woven materialsthrough fibers of the same materials by grafting method, providing thesurface properties of the fibers are same to that of the non-wovenmaterials. This method can be expressed by the following equation:Wt=D(S _(f) +S _(NW))where Wt is weight gain from grafting, S_(f) is surface area of fiberand S_(NW) is surface area of non-woven material. Only two unknowns arein the equation: D and S_(NW), which can be determined by twoindependent experiments.

The area determined by this method is the effective surface areacorresponding to each type reaction. Actually, any reactions whoseextents depend on the surface area of substrates can be used to for thismethod.

Conclusions:

Primary tests of grafting GMA on several polymer substrates show that PPis an ideal substrate for such grafting. The weight gain of grafting GMAon PP varies from 60% to 160%, depending on the shape of PP materials.The grafting effects are also confirmed by the FTIR spectra.Furthermore, based on grafting, a simply method has been proposed formeasuring surface area of non-woven materials (Table 6).

TABLE 6 Effects of grafting measured in weight gain UV Initial WeightTime Weight Gain Sample Name Substrate (hr) (g) (%) Co-g-GMA-032905Cotton woven fabric 0.6 0.1283 −1.8 Ny-g-GMA-032905 Nylon woven fabric0.6 0.1323 −0.7 PP-g-GMA-032905 PP non-woven 0.6 0.1179 1.7Co-g-GMA-033105 Cotton woven fabric 6 0.0954 −2.8 Ny-g-GMA-033105 Nylonwoven fabric 6 0.1446 −1.4 PP-g-GMA-033105 PP non-woven 6 0.0537 91Mac-PP-g-GMA-040805 Macopharma PP 6 0.0668 57 non-woven membraneNW-PP-g-GMA-040805 PP non-woven 6 0.0738 154 Fabric FB-PP-g-GMA-040805PP fibers 6 0.0789 85 FB-PET-g-GMA-040805 PET fibers 6 0.0517 4

Example 16 Binding of PrP^(c) by PGMA Fibers, Electro-Spun Web, PGMAGrafted and Padded PP Substrates

Purpose:

To determine the binding of PrP^(c) by PGMA fibers, electrospun web andGMA grafted and padded polypropylene non-wovens.

Procedure:

The materials tested were polyglycidylmethacrylate (PGMA) melt spunfibers, PGMA electro-spun webs, PGMA grafted and padded polypropylene(GMA-g-PP) nonwoven fabric. The substrates are samples from the Collegeof Textiles at North Carolina State University, MacoPharma PP175, andMacopharma PP 235. For each material, two replicas were prepared forprotein binding.

The samples were prepared in both weight and apparent area (the measuredarea of fabrics). The second method only applied to the samples withregular shapes, such as the padded MacoPharma membranes.

All the samples were chopped into small pieces. Each sample was thenimmersed into a conical tube (50 ml) containing 5 ml 1% normal hamsterbrain homogenate (HaBH) solution. Samples were then incubated on arocking platform for 30 minutes. After that, the samples were washedwith 10 ml sample buffer for three times of 10 minutes on the rockingplatform.

Results:

Sample weight, area and animation level which was determined byelemental analysis are shown in the following in Table 7 below

TABLE 7 Elemental Analysis of Samples.: Name N % Weight (g) PGMA-Fiber A0.1009 3.8% PGMA-Fiber B 0.1011 3.8% PGMA-g-PP A 0.1007 2.6% PGMA-g-PP B0.1009 2.6% PGMA-g-PP (Maco175) A 0.1011 1.5% PGMA-g-PP (Maco175) B0.1010 1.5% PGMA-p-PP1A 0.1015 Not available PGMA-p-PP1B 0.1007 Notavailable PGMA-p-PP2A 0.1007 Not available PGMA-p-PP2B 0.1012 Notavailable PGMA-p-PP3A 0.1012 Not available PGMA-p-PP3B 0.1007 Notavailable Blank-Nonwoven-PP A 0.1009 None Blank-Nonwoven-PP B 0.1011None Blank-Maco175A 0.1011 None Blank-Maco175B 0.1006 None 650MA 0.10160.6% 650MB 0.1011 0.6% Electro Spun A 0.1001 Not available Electro SpunB 0.1005 Not available Area (cm²) PGMA-p-PP (Maco235) 6A 4 × 4 Notavailable PGMA-p-PP (Maco235) 6B 4 × 4 Not available PGMA-p-PP (Maco235)7A 4 × 4 Not available PGMA-p-PP (Maco235) 7B 4 × 4 Not availablePGMA-p-PP (Maco235) 9A 4 × 4 Not available PGMA-p-PP (Maco235) 9b 4 × 4Not available PGMA-p-PP (Maco235) 10^(a) 4 × 4 Not available PGMA-p-PP(Maco235) 4 × 4 Not available 10B Blank-Maco235A 4 × 4 Not availableBlank-Maco235B 4 × 4 Not available

Based on the results from western blot both PGMA grafted PP and PGMApadded PP bind prion.

EQUIVALENTS

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionsthat have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed herein, optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

All references discussed herein are incorporated by reference. Oneskilled in the art will readily appreciate that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The present invention maybe embodied in other specific forms without departing from the spirit oressential attributes thereof. On the contrary, it is to be clearlyunderstood that resort may be had to various other embodiments,modifications, and equivalents thereof which, after reading thedescription herein, may suggest themselves to those skilled in the artwithout departing from the spirit of the present invention and/or thescope of the appended claims.

1. A device for filtering and separating at least one target agent froma sample that, in use, flows through said device, said devicecomprising: a casing that comprises an inlet and an outlet, and stackedlayers of resin-embedded membranes positioned internally of the casingbetween the inlet and the outlet, said resin-embedded membranescomprising first and second layers of porous nonwoven fabric that arebonded together with a plurality of resin particles impregnated thereinor sandwiched therebetween, said porous nonwoven fabric having poresizes greater than 10 μm, and the particles having a size of 40-200 μm,whereby sample flowing through the stacked layers of resin-embeddedmembranes in the casing causes the at least one target agent to attachto the porous nonwoven fabric, resin particles, or both, and be removedfrom the sample.
 2. The device of claim 1 wherein the resin particlesare porous, nonporous, or both.
 3. The device of claim 1 wherein theporous nonwoven fabric, the particles or both have a uniform or avariable pore size.
 4. The device of claim 2 wherein the particles havea pore size of about 0.001 μm to about 0.1 μm.
 5. The device of claim 1wherein the porous nonwoven fabric comprises natural fibers, syntheticfibers or both.
 6. The device of claim 2 wherein the resin particlescomprise a porous resin having interconnected pores with surface areasin the range of about 1-2 m²/g of dried resin to about 300 m²/g of driedresin.
 7. The device of claim 1 wherein the at least one target agentattaches to the resin particles, to the porous nonwoven fabric, or bothvia absorption, absorption, ion exchange, covalent bonds, hydrophobic,affinity interactions, formation of charged species, the attachment ofaffinity ligands, or a combination thereof.
 8. The device of claim 1wherein the porous nonwoven fabric, the resin particles, or both arefunctionalized on one or more internal and/or external surfaces with areactive group that interacts with and binds the target agent.
 9. Thedevice of claim 8 wherein the reactive group comprises a functionalgroup comprising epoxy, formyl, tresyl, hydroxysuccinimide esters,sulfonic acid, quaternary amines, carboxylic groups, primary amines,cyano, cyclohexyl, octyl, and octadecyl groups, epoxide, oxirane,N-hydroxysuccinimide esters, sulfonyl esters, imidazolyl carbamate,quaternary amines, carboxylic groups, dye ligand, affinity ligand,antigen-antibody, nucleic acid molecules, reactive groups for ionexchange, chelation, oxidation/reduction reactions, stearic exclusionreactions, catalysis reactions, hydrophobic reactions, or reverse phase,or a combination thereof.
 10. The device of claim 1 wherein the sampleis a blood sample and the at least one target agent comprises prions,viruses, bacteria, protozoa, and toxins, or a combination thereof. 11.The device of claim 1, wherein the resin particles comprise apolymethacrylate resin, a methacrylate resin, a modified resin, or acombination thereof.
 12. The device of claim 11, wherein the modifiedresin comprises TOYOPEARL™AMINO
 650. 13. The device of claim 6, whereinthe resin comprises a wet resin, a dry resin, or a combination thereof.14. The device of claim 9, wherein the resin particles comprise amodified resin, the porous nonwoven fabric comprises plasma treatedpolypropylene and the reactive group comprises a ligand having a primaryamine and a hydrophilic spacer containing polyethylene glycol units. 15.A device for filtering and separating at least one target agent from asample that, in use, flows through the device, said device comprising: acasing having an inlet and outlet, and one or more stacked layers ofporous nonwoven fabric positioned internally of the casing between theinlet and the outlet, said porous nonwoven fabric having pore sizeslarger than 10 μm, wherein the one or more stacked layers of porousnonwoven fabric comprise fibers functionalized on one or more surfacesthereof with a reactive group that interacts with and binds the targetagent, whereby sample flowing through the one or more stacked layers ofporous nonwoven fabric in the casing causes the at least one targetagent to bind to said fibers, thereby separating the at least one targetagent from the sample.
 16. A method of separating at least one targetagent from a sample comprising; (a) providing a sample potentiallycontaining one or more target agents; (b) providing a device accordingto claim 1; (c) subjecting the sample to the device; (d) attaching theat least one target agent to the particles, to the porous matrixmaterial, or both; and (e) separating the at least one target agent fromthe sample.
 17. The device of claim 1, wherein flow is induced throughthe device by gravity or a pump.
 18. The device of claim 1, wherein thestacked layers are welded together.
 19. The device of any claim 1,wherein the casing is formed of a plastic.
 20. The device of claim 5,wherein the porous nonwoven fabric comprises polypropylene or polyesterfibers.
 21. The device of claim 1 wherein the particles are choppedfibers.
 22. The device of claim 21 wherein the chopped fibers have alength to diameter ratio of about 1 μm to about 20 μm.
 23. The device ofclaim 1 further comprising one or more single layers of porous nonwovenfabric.
 24. The device according to claim 1, wherein said resin-embeddedmembranes consist of said first and second layers of porous nonwovenfabric, and said plurality of resin particles.
 25. A device forfiltering and separating at least one target agent from a sample that,in use, flows through said device, said device comprising: a casing thatcomprises an inlet and an outlet, and stacked layers of resin-embeddedmembranes positioned internally of the casing between the inlet and theoutlet, the stacked layers comprising between 1 and about 25 singlelayers of porous nonwoven fabric followed by between 1 and about 50layers of resin-embedded membranes, followed by between 1 and about 25single layers of porous nonwoven fabric, wherein said resin-embeddedmembranes comprise first and second layers of porous nonwoven fabricthat are bonded together with a plurality of resin particles impregnatedtherein or sandwiched therebetween, said porous nonwoven fabric havingpore sizes greater than 10 μm, and the resin particles having a size of40-200 μm, whereby sample flowing through the stacked layers ofresin-embedded membranes in the casing causes the at least one targetagent to attach to the porous nonwoven fabric, resin particles, or both,and be removed from the sample.